KR101829159B1 - Water soluble fluorescent or colored dyes - Google Patents

Abstract

상기 식에서, R^1a, R^1b, R^2a, R^2b, R^2c, R^2d, R^2e, R^2f, R^2g, R^2h, R²ⁱ, R^2j, R^2k, R^2l, R^2m, R²ⁿ, R^2o, R^2p, R^2q, R^2r 및 R^2s는 본원에 정의된 바와 같다. 제조와 관련된 방법 및 이러한 화합물의 용도가 또한 제공된다.Compounds useful as fluorescent or colored dyes are disclosed. The compounds have the following structure I, including stereoisomers, salts and tautomers thereof.
(I)

^{Wherein, R 1a, R 1b, R} 2a, R 2b, R 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n , R ^2o , R ^2p , R ^2q , R ^2r and R ^2s are as defined herein. Methods associated with manufacture and uses of such compounds are also provided.

Description

{WATER SOLUBLE FLUORESCENT OR COLORED DYES}

The present invention relates to novel fluorescent or colored dyes, their preparation and their use in various analytical methods.

There is a continuing and growing need for rapid, high-specific methods of detecting and quantifying chemical, biochemical and biological materials as analytes in research and diagnostic mixtures. Methods of measuring small quantities of nucleic acids, peptides, saccharides, pharmaceuticals, metabolites, microorganisms, ions, and other substances with diagnostic value are of particular value. Examples of such substances include drugs and poisons, drugs administered for therapeutic purposes, hormones, pathogenic microorganisms and viruses, peptides such as antibodies and enzymes, and nucleic acids, particularly those related to the disease state.

The presence of a particular analyte can often be determined by a binding method that utilizes a high degree of specificity that characterizes numerous biochemical and biological systems. Commonly used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. In these methods, the presence of a complex diagnostic value is typically indicated by the presence or absence of an observable "label" attached to one or more of the interacting materials. The particular labeling method chosen often indicates the availability and versatility of the particular system for detecting the analyte of interest. Preferred labels are inexpensive, safe, and can be effectively attached to such materials without significantly altering the critical binding affinity of a wide variety of chemical, biochemical, and biological materials. The label should provide a very distinctive signal, rarely found in nature, or preferably never found. The label should be stable and detectable in an aqueous system over a period of time in the range of a few months or less. Detection of the label is preferably rapid, sensitive, and reproducible without the need for expensive professional facilities or the need for special prevention to protect the individual. Quantification of the label is preferably relatively independent of the parameters, such as composition and temperature of the mixture to be analyzed.

A wide variety of markers have been developed, each with specific advantages and disadvantages. For example, radioactive labels are very versatile and can be detected at very low concentrations. However, these signs are expensive, dangerous, and their use requires sophisticated equipment and trained personnel. Thus, there is widespread interest in non-radioactive labels, in particular spectrophotometry, spin resonance, and luminescence techniques, and labels that can be observed by reactive substances, such as enzymes that produce such molecules.

Of particular interest are detectable labels using fluorescence spectroscopy because of the large number of such labels known in the art. In addition, the literature is filled with the synthesis of fluorescent labels that are derivatized and allow attachment of fluorescent labels to other molecules, and a number of such fluorescent labels are commercially available.

Cyanine dyes can be used to label biomolecules, including antibodies, DNA probes, avidin, streptavidin, lipids, biochemical analogs, peptides, and drugs, as well as DNA sequencing, DNA microarrays, And has been widely used for a variety of applications including blotting, flow cytometry analysis, and protein microarrays. Scientists, among other reasons, have found that cyanine dyes are 1) biocompatible; 2) a high molar extinction coefficient (about 10 ⁵ M ^- has a ^{1) - 1} ㎝; 3) is readily modified to meet a wide range of desired excitation and detection wavelengths (e.g., from about 500 to about 900 nm); 4) water-soluble groups and linking groups can be incorporated; 5) Because of their favorable fluorescence properties, they prefer to use cyanine dyes in biological applications. In particular, of the green spectrum of the visible spectrum, the Cy2 conjugate, which has maximum absorption / excitation at about 492 nm and emission at about 510 nm, is commonly used as an alternative to FITC because of its reduced sensitivity to pH changes. However, low fluorescence quantum yield, short fluorescence lifetime, trend towards light discoloration, and poor chemical stability of Cy2 have limited its use in chemistry and life sciences.

There is therefore a need in the art for water-soluble dyes and biomarkers that permit visual or fluorescent detection of biomolecules without previous illumination or chemical or enzymatic activation. Ideally, these dyes and biomarkers should be strongly colored or fluorescent and be available in a variety of colors and fluorescence wavelengths. The present invention meets this need and provides additional related benefits.

Briefly, the present invention relates generally to biomolecules and other analytes, as well as to compounds useful as water-soluble fluorescent or colored dyes and probes to enable visual detection of their reagents for manufacture. A method of visually detecting biomolecules and measuring the size of biomolecules is also described. The water-soluble fluorescent or colored dyes of the present invention are strongly color and / or fluorescent and can be easily observed by visual inspection or other means. In some embodiments, the compound can be observed without previous illumination or chemical or enzymatic activation. Advantageously, embodiments of the dye have a maximum absorbance in the range of about 468 nm to about 508 nm and a maximum emission in the range of about 495 nm to about 525 nm. For example, in certain embodiments, the dye has a maximum absorbance at about 490 nm and a maximum emission at about 505 nm. The dyes are therefore ideal for use in a variety of analytical methods. As described herein, visually detectable biomolecules of various colors can be obtained by appropriate selection of the dyes.

Thus, in one embodiment, there is provided a compound having the structure I below, or a salt, stereoisomer or tautomer thereof.

(I)

^{Wherein, R 1a, R 1b, R} 2a, R 2b, R 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n , R ^2o , R ^2p , R ^2q , R ^2r , R ^2s and R ^2t are as defined herein.

In another embodiment, there is provided an analyte molecule comprising a covalent bond to a compound having the structure I < 1 >, or a salt, stereoisomer or tautomer thereof.

<Formula I '

^{Wherein, R 1a, R 1b, R} 2a, R 2b, R 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n ^{, R 2o, R 2p, R} 2q, R 2r, R 2s and R ^2t is as defined herein, ^{R 2a, R 2b, R 2c} , R 2d, R 2e, R 2f, R 2g, R 2h, R ^{At least one of the 2i} , R ^2j , R ^2k , R ^2l , R ^2m , R ²ⁿ , R ^2o , R ^2p , R ^2q , R ^2r , R ^2s or R ^2t is an analyte molecule or a linker thereto.

In another embodiment, a method of dyeing a sample is provided; The method comprises adding to the sample a representative compound as described herein in an amount sufficient to cause an optical reaction when illuminating the sample at the appropriate wavelength.

In another embodiment,

(a) providing a representative compound as described herein;

(b) detecting the compound by its visual characteristics

A method for detecting a biomolecule visually.

Another disclosed method is

(a) mixing any of the disclosed compounds with one or more biomolecules;

(b) detecting the compound by its visual characteristics

And a method for visually detecting a biomolecule.

Another embodiment relates to a composition comprising any one of the disclosed compounds and one or more biomolecules. Use of such a composition in an assay method for detecting one or more biomolecules is also provided.

These and other aspects of the invention will become apparent upon reference to the following detailed description.

1 shows the ¹ H NMR spectrum of Compound 2 in CDCl ₃ .
Figure 2 shows a representative total diode array chromatogram (215-500 nm) of compound 2 at 2 주입 injection volume. The system used was a Waters liquid Qwerty UHPLC system with a 2.1 mm x 50 mm Acquity BEH-C18 column maintained at 45 &lt; 0 &gt; C.
Figure 3 shows the background-subtracted mass spectrum of the brominated anthracene derivative 2 product. Expected Mass: 617.8.
4 shows the ¹ H NMR spectrum of compound 3 in CDCl ₃ .
Figure 5 shows a representative total diode array chromatogram (215-500 nm) of compound 3 at 2 [mu] l injection volume. The system used was a Waters AccuQuid UHPLC system equipped with a 2.1 mm x 50 mm Axuch BEH-C18 column maintained at 45 &lt; 0 &gt; C.
Figure 6 shows the background-subtracted mass spectrum of compound 3. Expected mass 929.1.
Figure 7 depicts representative chromatograms at 488 nm of Compound 5 (crude 5'-NH3- (HEG) -AQ6- (HEG) -3 'sequence) at 10 μl injection volume. The system used was a Waters AccuQuid UHPLC system equipped with a 2.1 mm x 50 mm Axuch BEH-C18 column maintained at 45 &lt; 0 &gt; C.
Figure 8 shows the background-subtracted mass spectrum of compound 5 (crude 5'-NH3- (HEG) -AQ6- (HEG) -3 'sequence). Method using electrospray ionization in negative mode, ^- 1, ^- 2 and ^- shows the three charge states.

In the following description, specific specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, one of ordinary skill in the art will appreciate that the present invention may be practiced without these details.

It is to be understood that the word "comprises" and variations thereof, such as "comprising" and "comprising ", in an open, inclusive sense, Quot; and " not limited to. "

Reference throughout this specification to "one embodiment" or "one embodiment " means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, certain features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

"Amino" refers to the group -NH ₂ .

"Carboxyl" refers to a group -CO ₂ H.

"Cyano" refers to a -CN group.

"Formyl" refers to the group -C (= O) H.

"Hydroxy" or "hydroxyl"

"Imino" refers to the = NH group.

"Nitro" refers to a -NO ₂ group.

"Oxo" refers to the group = O substituent.

"Sulfhydryl" refers to the -SH group.

"Thioxo" refers to the = S group.

"Alkyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing from 1 to 12 carbon atoms (C ₁ -C ₁₂ alkyl), preferably from 1 to 8 carbon atoms (C ₁ -C Saturated or unsaturated (i.e. containing one or more double and / or triple bonds), having from 1 to 6 carbon atoms (C ₁ -C ₈ alkyl) or one to six carbon atoms (C ₁ -C ₆ alkyl) (Iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, . Unless specifically stated otherwise herein, alkyl groups are optionally substituted.

"Alkylene" or "alkylene chain" refers to a straight or branched chain linking group that connects the remainder of the molecule to a group of substituents and is saturated or unsaturated (i.e. containing one or more double and / or triple bonds) Refers to a linear or branched divalent hydrocarbon chain having from 1 to 12 carbon atoms and includes, for example, methylene, ethylene, propylene, n-butylene, ethenylene, prophenylene, Butynylene. The alkylene chain is attached to the rest of the molecule through a single or double bond and to a group of substituents through a single or double bond. The rest of the molecule and the point of attachment of the alkylene chain to the group of substituents may be through one carbon in the chain or any two carbons. Unless specifically stated otherwise herein, the alkylene chain is optionally substituted.

"Alkoxy" refers to a group of the formula -OR _a wherein R _a is an alkyl group as defined above containing from 1 to 12 carbon atoms. "Hydroxylalkoxy" is an alkoxy moiety containing at least one hydroxyl substituent. "Aminoalkoxy" is an alkoxy moiety containing one or more amino substituents. Unless specifically stated otherwise herein, alkoxy, hydroxylalkoxy and / or aminoalkoxy groups are optionally substituted.

"Alkylamino" refers to a group of the formula -NHR _a or -NR _a R _a wherein each R _a is independently an alkyl group, as defined above, containing from 1 to 12 carbon atoms. Unless specifically stated otherwise herein, alkylamino groups are optionally substituted.

"Alkyl ether" refers to any alkyl group as defined above wherein at least one carbon-carbon bond is replaced by a carbon-oxygen bond. The carbon-oxygen bond may be on the terminal end (as in an alkoxy group) or the carbon oxygen bond may be internal (i.e., C-O-C). The alkyl ether contains one or more carbon oxygen bonds, but may contain more than one (i.e., "polyalkyl ether"). For example, polyethylene glycol (PEG), which is a polyalkyl ether, is included within the meaning of an alkyl ether. "Hydroxyl polyalkyl ether" refers to a polyalkyl ether comprising at least one hydroxyl substituent. "Aminopolyalkyl ether" refers to a polyalkyl ether containing one or more amino substituents. Unless specifically stated otherwise specifically herein, alkyl ether, polyalkyl ether, hydroxylpolyalkyl ether and / or aminopolyalkyl ether groups are optionally substituted.

"Alkylene &lt; / RTI &gt; ether" refers to any alkylene group as defined above wherein one or more carbon-carbon bonds are replaced by carbon-oxygen bonds. The carbon-oxygen bond may be on the terminal end (as in an alkoxy group) or the carbon oxygen bond may be internal (i.e., C-O-C). Alkylene ethers include one or more carbon oxygen bonds, but may include more than one. For example, polyethylene glycol (PEG) is included within the meaning of an alkylene ether. Unless specifically stated otherwise herein, alkylene ether groups are optionally substituted.

Refers to the group -RP (= O) (R _a ) R _b , wherein R is an alkylene group and R _a is OH, O ^- or OR _c ; R _b is -O alkyl or -O alkyl ether, wherein R _c is a counterion (e.g., Na +, etc.). Unless specifically stated otherwise herein, alkylphospho groups may be optionally substituted. For example, in certain embodiments, the alkyl or alkyl ether moiety (R _b ) in the alkylphospho group is selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Alkyl ether or thiophosphoalkyl ether, which substituent is also optionally substituted.

"Oalkylphospho" is an alkylphospho group attached to the remainder of the molecule through an oxygen atom. Unless specifically stated otherwise herein, the O alkylphospho group is optionally substituted. For example, in certain embodiments, the alkyl, alkyl ether, or polyalkyl ether moiety (R _b ) in the O alkylphospho group is selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, Alkyl, alkenyl, alkynyl, alkynyl, haloalkyl, haloalkyl, haloalkyl, haloalkyl, haloalkyl,

Refers to the group -RP (= O) (R _a ) R _b wherein R is an alkylene ether group and R _a is OH, O ^- or OR _c ; R _b is -O alkyl or -O alkyl ether, wherein R _c is a counterion (e.g., Na +, etc.). Unless specifically stated otherwise herein, the alkyl ether phospho group is optionally substituted. For example, in certain embodiments, the alkyl or alkyl ether moiety (R _b ) in the alkyl ether phospho group is selected from the group consisting of hydroxyl, amino, sulfhydryl or phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Or a thiophosphoalkyl ether, which substituent is optionally also optionally substituted.

An " O alkyl ether phospho "is an alkyl ether phospho group connected to the remainder of the molecule through an oxygen atom. Unless specifically stated otherwise herein, the O alkyl ether phospho group is optionally substituted. For example, in certain embodiments, the alkyl or alkyl ether moiety (R _b ) in the O alkyl ether phospho group is selected from the group consisting of hydroxyl, amino, sulfhydryl or phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, A phosphoalkyl ether, or a thiophosphoalkyl ether, which substituent is also optionally substituted.

"Alkylthio phosphonate" are _{-RP (= R a) (R} b) refers to the group R _c, where R is an alkylene group, and R _a is O or S, R _b is OH, O ^-, S ^- , OR _d or SR _d ; R _c is -O alkyl or -O alkyl ether wherein R _d is a counterion (e.g. Na + etc.), with the proviso that R _a is S or R _b is S ^- or SR _d ; Or R _a is S and R _b is S ^- or SR _d . Unless specifically stated otherwise herein, alkylthiophospho groups are optionally substituted. For example, in certain embodiments, the alkyl or alkyl ether moiety in the alkylthiophosphoryl group is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Thiophosphoalkyl &lt; / RTI &gt; ether, which substituent is also optionally substituted.

An "O alkylthiophosphorus" is an alkylthiophosphorus group linked to the remainder of the molecule through an oxygen atom. Unless specifically stated otherwise herein, the O alkylthiophospho group is optionally substituted. For example, in certain embodiments, the alkyl or alkyl ether moiety in the O alkylthiophospho group is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Or thiophosphoalkyl ether, which substituent is optionally also optionally substituted.

"Alkyl thio ether phosphonate" are _{-RP (= R a) (R} b) refers to the group R _c, where R is an alkylene ether group, and R _a is O or S, R _b is OH, O ^-, S ^- , OR _d or SR _d ; R _c is -O alkyl or -O alkyl ether wherein R _d is a counterion (e.g. Na + etc.), with the proviso that R _a is S or R _b is S ^- or SR _d ; Or R _a is S and R _b is S ^- or SR _d . Unless specifically stated otherwise herein, the alkyl ether thiophospho group is optionally substituted. For example, in certain embodiments, the alkyl or alkyl ether moiety in the alkyl ether thiophosphoryl group is selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Or thiophosphoalkyl ether, which substituent is optionally also optionally substituted.

Is an alkyl ether thiophosphorus group linked to the remainder of the molecule through an oxygen atom. Unless specifically stated otherwise specifically herein, the O alkyl ether thiophospho group may be optionally substituted. For example, in certain embodiments, the alkyl or alkyl ether moiety in the O alkyl ether thiophosphoric group is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Ether or thiophosphoalkyl ether, which substituent is also optionally substituted.

"Amidyl" refers to the radical -NR _a R _b where R _a and R _b are independently H, alkyl or aryl. Unless specifically stated otherwise herein, the amide group is optionally substituted.

"Aryl" refers to a hydrocarbon ring group comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For purposes of the present invention, the aryl group may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may include fused or bridged ring systems. The aryl group may be substituted with at least one group selected from the group consisting of acenylene, acenaphthylene, acenaphthylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as- , Phenanthrene, playaden, pyrene, and triphenylene. &Lt; / RTI &gt; Unless specifically stated otherwise specifically herein, the term "aryl" or the prefix "ar-" (eg, in "aralkyl") is intended to include an optionally substituted aryl group.

"Aryloxy" refers to a group of the formula -OR _a wherein R _a is an aryl moiety as defined above, such as phenoxy, and the like. Unless specifically stated otherwise herein, the aryloxy group is optionally substituted.

"Aralkyl" refers to a group of the formula -R _b -R _c , wherein R _b is an alkylene chain as defined above and R _c is one or more aryl groups as defined above, Diphenylmethyl and the like. Unless specifically stated otherwise specifically herein, aralkyl groups are optionally substituted.

"O Aralkyl" is an aralkyl group that is linked to the remainder of the molecule through an oxygen linkage. "ODMT" refers to dimethoxytrityl linked to the remainder of the molecule through the O atom. Unless specifically stated otherwise herein, the O aralkyl group is optionally substituted.

"Cyanoalkyl" refers to an alkyl group containing one or more cyano substituents. One or more -CN substituents may be on a primary, secondary or tertiary carbon atom. Unless specifically stated otherwise herein, cyanoalkyl groups are optionally substituted.

"Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting solely of carbon and hydrogen atoms, containing from 3 to 15 carbon atoms, preferably from 3 to 10 carbon atoms , Fused or bridged ring systems, saturated or unsaturated and attached to the remainder of the molecule by a single bond. Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The polycyclic cycloalkyl group includes, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo [2.2.1] heptanyl and the like. Unless specifically stated otherwise herein, the cycloalkyl group is optionally substituted.

"Cycloalkylalkyl" refers to a group of the formula -R _b R _d , wherein R _b is an alkylene chain as defined above and R _d is a cycloalkyl group as defined above. Unless specifically stated otherwise specifically herein, a cycloalkylalkyl group is optionally substituted.

"Multicycle" refers to any molecule having more than one ring. The ring may be a fused spirocyclic or may be separated by one or more atoms (e.g., via a bicyclic linker).

"Spirocyclic" refers to a multicyclic molecule in which two rings share a single carbon atom.

"Fused" refers to any of the ring structures described herein fused to the ring structure present in the compounds of the present invention. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure that is part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced by a nitrogen atom.

"Halo" or "halogen" refers to bromo, chloro, fluoro or iodo.

"Haloalkyl" means an alkyl group as defined above, which is substituted by one or more halo groups as defined above, for example trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2 - trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl and the like. Unless specifically stated otherwise herein, haloalkyl groups are optionally substituted.

"Heterocyclyl" or "heterocyclic ring" refers to a stable 3- to 18-membered non-aromatic ring group of 2 to 12 carbon atoms and consisting of 1 to 6 heteroatoms selected from the group consisting of nitrogen, . Unless specifically stated otherwise specifically herein, a heterocyclyl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused or bridged ring system; The nitrogen, carbon or sulfur atom in the heterocyclyl group may optionally be oxidized; The nitrogen atom may optionally be quaternized; The heterocyclyl group may be partially or fully saturated. Examples of such heterocyclyl groups are dioxolanyl, thienyl [1,3] dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinyl, tetrahydropyranyl, , Pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo- thiomorpholinyl, and But are not limited to, 1,1-dioxo-thiomorpholinyl. Unless specifically stated otherwise herein, the heterocyclyl group is optionally substituted.

Refers to a heterocyclyl group as defined above containing one or more nitrogen atoms wherein the point of attachment of the heterocyclyl group to the remainder of the molecule is through a nitrogen atom in the heterocyclyl group to be. Unless specifically stated otherwise herein, the N-heterocyclyl group is optionally substituted.

"Heterocyclylalkyl" refers to a group of the formula -R _b R _e , wherein R _b is an alkylene chain as defined above, R _e is a heterocyclyl group as defined above, and heterocyclyl In the case of a nitrogen-containing heterocyclyl, the heterocyclyl may be attached to an alkyl group at the nitrogen atom. Unless specifically stated otherwise herein, a heterocyclylalkyl group is optionally substituted.

"Heteroaryl" means a 5- to 14-membered ring system comprising 1 to 6 heteroatoms selected from the group consisting of hydrogen atoms, 1 to 13 carbon atoms, nitrogen, oxygen and sulfur, and one or more aromatic rings. Quot; For purposes of the present invention, a heteroaryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused or bridged ring system; The nitrogen, carbon or sulfur atom in the heteroaryl group may optionally be oxidized; The nitrogen atom may optionally be quatemized. Examples include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo [b] [1 , 4] dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranyl, benzofuranyl, benzofura Nonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo [4,6] imidazo [1,2-a] pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl , Furanyl, furanyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridyl Oxydopyrimidinyl, 1-oxydopyridazinyl, 1-oxydopyridazinyl, 1-oxopyridazinyl, 1-oxopyridazinyl, Phenyl- Pyridyl, pyridinyl, pyrimidinyl, pyridazinyl, quinazolinyl, pyrazinyl, pyrimidinyl, pyrimidinyl, pyrimidinyl, pyrimidinyl, pyrimidinyl, (I. E., Thienyl) may be optionally substituted with one or more substituents selected from the group consisting of lower alkyl, lower alkoxy, lower alkoxy, lower alkoxy, lower alkoxy, lower alkoxy, lower alkoxy, But are not limited thereto. Unless specifically stated otherwise herein, the heteroaryl group is optionally substituted.

Refers to a heteroaryl group as defined above containing one or more nitrogen atoms wherein the point of attachment of the heteroaryl group to the remainder of the molecule is through the nitrogen atom in the heteroaryl group. Unless specifically stated otherwise herein, the N-heteroaryl group is optionally substituted.

"Heteroarylalkyl" refers to a group of the formula -R _b R _f , wherein R _b is an alkylene chain as defined above and R _f is a heteroaryl group as defined above. Unless specifically stated otherwise herein, heteroarylalkyl groups are optionally substituted.

"Hydroxylalkyl" refers to an alkyl group containing one or more hydroxyl substituents. One or more -OH substituents may be on a primary, secondary or tertiary carbon atom. Unless specifically stated otherwise herein, the hydroxyalkyl group is optionally substituted.

"Hydroxylalkyl ether" refers to an alkyl ether group comprising at least one hydroxyl substituent. One or more -OH substituents may be on a primary, secondary or tertiary carbon atom. Unless specifically stated otherwise herein, the hydroxyalkyl ether group is optionally substituted.

"Phosphate" refers to the group -OP (= O) (R _a ) R _b wherein R _a is OH, O ^- or OR _c ; R _b is OH, O ^- or OR _c , an additional phosphate group (as in diphosphate and triphosphate), thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, wherein R _c is a counterion (e.g., Na +, etc.). Unless specifically stated otherwise herein, the phosphate group is optionally substituted.

Refers to the group -P (= O) (R _a ) R _b wherein R _a is OH, O ^- or OR _c ; R _b is OH, O ^- or OR _c , a phosphate group (as in diphosphate and triphosphate), thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, wherein R _c is a counter ion (e.g., Na +, etc.). Unless specifically stated otherwise herein, the phospho group is optionally substituted.

"Phosphoalkyl" refers to the group -P (= O) (R _a ) R _b wherein R _a is OH, O ^- or OR _c ; R _b is -O alkyl, wherein R _c is a counterion (e.g., Na +, etc.). Unless specifically stated otherwise herein, the phosphoalkyl group is optionally substituted. For example, in certain embodiments, the alkyl moiety in the phosphoalkyl group is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfhydryl or phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Ether, which substituent is optionally substituted.

"O-Phosphoalkyl" is a phosphoalkyl group linked to the remainder of the molecule through an oxygen atom. Unless specifically stated otherwise herein, the O-phosphoalkyl group is optionally substituted. For example, in certain embodiments, the alkyl moiety of the O-phosphoalkyl group is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfhydryl or phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Alkyl ether, and the substituent is optionally substituted.

"Phosphoalkyl ether" refers to the group -P (= O) (R _a ) R _b wherein R _a is OH, O ^- or OR _c ; R _b is an -O alkyl ether (including a polyether, such as polyethylene oxide ether and the like), wherein R _c is a counter ion (e.g., Na + etc.). Unless specifically stated otherwise herein, the phosphoalkyl ether group is optionally substituted. For example, in certain embodiments, the alkyl ether moiety in the phosphoalkyl ether group is selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, phospho, thiophospho, thiophosphate, phosphoalkyl, thiophosphoalkyl, Lt; / RTI &gt; is optionally substituted with one or more of the following groups: a phosphoalkyl ether or a thiophosphoalkyl ether, which substituent is optionally substituted.

"O-Phosphoalkyl ether" is a phosphoalkyl ether group linked to the remainder of the molecule through an oxygen atom. Unless specifically stated otherwise herein, the O-phosphoalkyl ether group is optionally substituted. For example, in certain embodiments, the alkyl ether moiety in the O-phosphoalkyl ether group is a hydroxyl, amino, sulfhydryl, phosphate, phospho, thiophospho, thiophosphate, phosphoalkyl, thiophosphoalkyl , A phosphoalkyl ether or a thiophosphoalkyl ether, the substituent being optionally substituted.

"Phosphoramidite" refers to the group -OP (OR ^a ) (NR ^b ₂ ) wherein R ^a is alkyl and each R ^b is independently H or alkyl. Unless specifically stated otherwise herein, the phosphoramidite group is optionally substituted.

"Activating phosphorus" refers to any moiety capable of reacting with the nucleophile, including phosphorus, for example, reacting with a nucleophile at a phosphorus atom. For example, moieties and phosphoramidites containing a P-halogen bond are included within the definition of moieties that are active. Unless specifically stated otherwise herein, the activating group is optionally substituted.

"Protected hydroxyl" refers to a hydroxyl moiety in which H is reversibly replaced by a protecting group. Protecting groups are well known in the art. In certain embodiments, the protected hydroxyl will be an ether (e.g., alkoxy, arylalkyloxy, or aryloxy). A non-limiting example of a protected hydroxyl is dimethoxytrityl ether. Other protected hydroxyl moieties are well known in the relevant art. Unless specifically stated otherwise herein, the protected hydroxyl group is optionally substituted.

"Sulfhydrylalkyl" refers to an alkyl group containing one or more sulfhydryl substituents. One or more -SH substituents may be on a primary, secondary or tertiary carbon atom. Unless specifically stated otherwise herein, sulfhydrylalkyl groups are optionally substituted.

"Sulfhydrylalkyl ether" refers to an alkyl ether group comprising at least one sulfhydryl substituent. One or more -SH substituents may be on a primary, secondary or tertiary carbon atom. Unless specifically stated otherwise herein, the sulfhydrylalkyl ether group is optionally substituted.

"Sulfonate" refers to the group -OS (O) ₂ R _a , wherein R _a is alkyl or aryl. Unless specifically stated otherwise herein, the sulfonate group is optionally substituted.

"Thioalkyl" refers to a group of the formula -SR _a , wherein R _a is an alkyl group as defined above containing from 1 to 12 carbon atoms. Unless specifically stated otherwise herein, the thioalkyl group is optionally substituted.

"Thiophosphate" is _{-OP (= R a) (R} b) refers to the group R _c, where R _a is O or S, R _b is OH, O ^-, S ^-, OR _d or _d and SR; R _c is OH, O ^- , OR _d , phosphate group, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, wherein R _d is a counter ion (e.g. Na + Etc., provided that R _a is S or R _b is S ^- or SR _d ; Or R _a is S and R _b is S ^- or SR _d . Unless specifically stated otherwise herein, the thiophosphate group is optionally substituted.

"Thio phosphonate" is _{-P (= R a) (R} b) refers to the group R _c, where R _a is O or S, R _b is OH, O ^-, S ^-, OR _d or _d and SR; R _c is OH, O ^- , OR _d , phosphate group, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, wherein R _d is a counter ion (e.g. Na + Etc., provided that R _a is S or R _b is S ^- or SR _d ; Or R _a is S and R _b is S ^- or SR _d . Unless specifically stated otherwise herein, the thiophosphorus group is optionally substituted.

"Thio phosphonate alkyl" and refers to the group _{-P (= R a) (R} b) R c, where R _a is O or S, R _b is ^{OH, O -, S -,} OR d and _d, or SR ; R _c is -O alkyl, wherein R _d is a counterion (e.g. Na + etc.), with the proviso that R _a is S or R _b is S ^- or SR _d ; Or R _a is S and R _b is S ^- or SR _d . Unless specifically stated otherwise herein, the thiophosphoalkyl group is optionally substituted. For example, in certain embodiments, the alkyl moiety of the thiophosphoalkyl group is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Alkyl ether, and the substituent is optionally substituted.

"O Thiophosphoalkyl" is a thiophosphoalkyl group linked to the remainder of the molecule through an oxygen atom. Unless specifically stated otherwise herein, the O-thiophosphoalkyl group is optionally substituted. For example, in certain embodiments, the alkyl moiety in the O-thiophosphoalkyl group is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, Lt; / RTI &gt; is optionally substituted with one or more of the following groups:

"Thio phosphonate alkyl ether" is _{-P (= R a) (R} b) refers to the group R _c, where R _a is O or S, R _b is OH, O ^-, S ^-, OR _d or SR _d ego; R _c is an -O alkyl ether, wherein R _d is a counterion (e.g. Na + etc.), with the proviso that R _a is S or R _b is S ^- or SR _d ; Or R _a is S and R _b is S ^- or SR _d . Unless specifically stated otherwise herein, the thiophosphoalkyl ether group is optionally substituted. For example, in certain embodiments, the alkyl ether moiety in the thiophosphoalkyl group is selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, phospho, thiophospho, thiophosphate, phosphoalkyl, thiophosphoalkyl, Lt; / RTI &gt; is optionally substituted with one or more of the following groups: a phosphoalkyl ether or a thiophosphoalkyl ether, which substituent is optionally substituted.

"O Thiophosphoalkyl ether" is a thiophosphoalkyl ether group linked to the remainder of the molecule through an oxygen atom. Unless specifically stated otherwise herein, the O thiophosphoalkyl ether group is optionally substituted. For example, in certain embodiments, the alkyl ether moiety in the O-thiophosphoalkyl group is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfhydryl, phosphate, phospho, thiophospho, thiophosphate, , A phosphoalkyl ether or a thiophosphoalkyl ether, the substituent being optionally substituted.

The term "substituted" as used herein refers to any of the foregoing groups (e.g., alkyl, alkylene, alkoxy, alkylamino, alkyl ether, polyalkylether, hydroxylpolyalkylether, aminopolyalkylether, alkyl Alkylaryl, arylalkyl, arylalkyl, Oaralkyl, cyanoalkyl, cycloalkyl, cycloalkyl, cycloalkylalkyl, arylalkyl, Alkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, heteroarylalkyl, hydroxylalkyl, aminoalkyl, hydroxylalkyl ether, phospho, , Phosphorous alkyl ether, phosphoramidite, activated phosphorus, protected hydroxyl, sulfhydrylalkyl, sulfhydrylalkyl ether, sulfonate, thioalkyl, thiophospho, thiophosphoalkyl and / or thiophos &Lt; / RTI &gt; And wherein one or more hydrogen atoms are halogen atoms, such as F, Cl, Br, and I; An oxygen atom in a group such as a hydroxyl group, an alkoxy group, and an ester group; A sulfur atom in a group such as a thiol group, a thioalkyl group, a sulfone group, a sulfonyl group, and a sulfoxide group; Nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; A silicon atom in a group such as a trialkylsilyl group, a dialkylarylsilyl group, an alkyldiarylsilyl group, and a triarylsilyl group; And non-hydrogen atoms, such as, but not limited to, other heteroatoms in the various other groups. "Substituted" also refers to any of the foregoing groups, wherein at least one hydrogen atom is replaced by a heteroatom such as oxygen in the oxo, carbonyl, carboxyl, and ester groups; And higher order bonds (e.g., double- or triple bonds) to nitrogen in the group such as imine, oxime, hydrazone, and nitrile. For example, "substituted" includes any of the foregoing groups in which one or more hydrogen atoms are replaced by -NR _g R _h , -NR _g C (= O) R _h , -NR _g C _{NR g R h, -NR g C} (= O) OR h, -NR g SO 2 R h, -OC (= O) NR g R h, -OR g, -SR g, -SOR g, -SO 2 R _g , -OSO ₂ R _g , -SO ₂ OR _g , = NSO ₂ R _g , and -SO ₂ NR _g R _h . "Substituted" also means any of the above groups, in which one or more hydrogen atoms are _{-C (= O) R g,} -C (= O) OR g, -C (= O) NR g R h , -CH ₂ SO ₂ R _g , -CH ₂ SO ₂ NR _g R _h . R _g and R _h are the same or different and are independently selected from the group consisting of hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, Heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. "Substituted" means any of the above groups wherein one or more of the hydrogen atoms may be replaced by an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, Is meant a bond to a bond to a thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / . In addition, each of the above substituents may also be optionally substituted with one or more of the above substituents.

"Fluorescence" refers to a molecule capable of absorbing light of a particular frequency and emitting light of a different frequency. Fluorescence is widely known to the ordinarily skilled artisan.

"Colored" refers to a molecule that absorbs light within a colored spectrum (i.e., red, yellow, blue, etc.).

A "linker" is a linker having one or more atoms, such as carbon, oxygen, nitrogen, sulfur, or sulfur, which connects a portion of the molecule to another moiety of the same molecule or to a different molecule, moiety, or solid support And adjacent chains of the combination. Linkers can link molecules through covalent bonds or other means, such as ionic or hydrogen bond interactions.

For purposes of the present invention, the term "biomolecule" refers to any of a variety of biological materials, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, More specifically, the term includes without limitation, RNA, DNA, oligonucleotides, modified or derivatized nucleotides, enzymes, receptors, prions, receptor ligands (including hormones), antibodies, antigens and toxins, as well as bacteria, Cells, and tissue cells. The visually detectable biomolecules of the present invention (i.e., the compounds of structure I having biomolecules linked thereto) can be prepared by reacting a biomolecule with any available atom or functional group such as amino, With a compound having a reactive group that enables attachment of the biomolecule to the compound via a hydroxyl, carboxyl, or sulfhydryl group.

The terms "visual" and "visually detectable" are used herein to refer to materials that can be observed by visual inspection, without previous illumination, or chemical or enzymatic activation. Such visually detectable materials absorb and emit light in the region of the spectrum in the range of about 300 to about 900 nm. Preferably, this material is intensely colored Castle, preferably between about 40,000 M ^{- 1} ㎝ ^-1 or more, more preferably and more preferably, about 50,000 M ^-1 ㎝ ^-1, greater than or equal to about 60,000 M ^{- 1} ㎝ ^-1 or higher, more and more preferably, from about 70,000 M ^-1 ㎝ ^-1 or greater, and most preferably between about 80,000 M ^{- 1} has a molar extinction coefficient ㎝ ^-1 or more. The biomolecules of the present invention can be detected by observation with the aid of an optically-based detection apparatus including an absorption spectrophotometer, a transmission light microscope, a digital camera and a scanner, either visually or without limitation. Visually detectable materials are not limited to those that emit and / or absorb light in the visible spectrum. Materials that emit and / or absorb light in the ultraviolet (UV) region (about 10 nm to about 400 nm), the infrared (IR) region (about 700 nm to about 1 mm), and other regions of the electromagnetic spectrum, Or absorbing materials are also included within the scope of "visually detectable" materials.

For purposes of the present invention, the term "photostable visible dye" refers to a chemical moiety that is visually detectable, as defined herein, and which is not significantly altered or degraded upon exposure to light. Preferably, the light stable visible dye does not exhibit significant discoloration or degradation after exposure to light for more than one hour. More preferably, the visible dye is stable after exposure to light for at least 12 hours, more preferably at least 24 hours, even more preferably at least one week, and most preferably at least one month. Non-limiting examples of photostable visible dyes suitable for use in the compounds and methods of the present invention include azo dyes, thioindigo dyes, quinacridone pigments, dioxazine, phthalocyanine, perinone, diketopyrrolopyrrole, quinophthalone, and Triarylcarbonium.

The visually detectable biomolecules of the present invention are useful in a wide variety of biochemical and biomedical applications where the presence, location, or quantity of a particular biomolecule needs to be determined. Thus, in another aspect, the present invention provides a method for detecting a biomolecule comprising: (a) providing a visually detectable biomolecule comprising a compound of structure I linked to a biomolecule in a biological system; (b) detecting the biomolecule by its visual characteristic. The present invention also provides a method for visually detecting a biomolecule. For the purpose of the present invention, the phrase "detecting a biomolecule by its visual characteristics" means that the biomolecule does not undergo illumination or chemical or enzymatic activation, either visually or without limitation, an absorption spectrophotometer, &Lt; / RTI &gt; and a scanner. &Lt; / RTI &gt; Densitometers can be used to quantify the amount of visually detectable biomolecules present. For example, the relative quantity of biomolecules in two samples can be determined by measuring the relative optical density. If the stoichiometry of the dye molecule per biomolecule is known and the extinction coefficient of the dye molecule is known, the absolute concentration of the biomolecule can also be determined from the measurement of the optical density. As used herein, the term "biological system" is used to refer to any solution or mixture comprising one or more biomolecules in addition to visually detectable biomolecules. Non-limiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoresis gels, assay mixtures, and hybridization reaction mixtures.

Refers to any of a number of small particles useful for adhering to a compound of the present invention, including, but not limited to, glass beads, magnetic beads, polymeric beads, non-polymeric beads and the like. In certain embodiments, the microparticles comprise polystyrene, such as polystyrene beads.

Embodiments of the invention disclosed herein also contemplate inclusion of all compounds of the isotope-labeled structure I by having at least one atom replaced by an atom having a different atomic mass or mass number. Disclosed Examples of isotopes that can be incorporated into compounds of hydrogen, isotopes of carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as ^{2 H, 3 H, 11 C} , 13 C, 14 C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ³⁶ Cl, ¹²³ I and ¹²⁵ I, respectively.

The isotopically-labeled compounds of structure I are generally prepared by conventional techniques known to the ordinarily skilled artisans or by processes analogous to those described below and in the appropriate isotopically-labeled Can be prepared in the following examples using reagents.

"Stable compound" and "stable structure" are intended to represent compounds that are sufficiently robust to isolate the reaction mixture to a useful degree of purity and to formulate it into an effective therapeutic agent.

"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the instant disclosure encompasses instances where the event or circumstance occurs and instances in which it does not. For example, "optionally substituted alkyl" means that the alkyl group may be substituted or unsubstituted, and that the context includes both substituted alkyl groups and unsubstituted alkyl groups.

"Salts" include both acid and base addition salts.

"Acid addition salts" include, but are not limited to, inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like and inorganic acids such as acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, , Benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, Gluconic acid, glutaric acid, glutaric acid, glutaric acid, 2-hydroxypropionic acid, tartaric acid, tartaric acid, glycolic acid, But are not limited to, oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, , 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, But are not limited to, hydrochloric acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, &Lt; / RTI &gt; such as, but not limited to, organic acids such as, but not limited to,

"Base addition salt" refers to such salts prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine , Triethylamine, tripropylamine, diethanolamine, ethanolamine, decanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine , Choline, betaine, venetamine, benzathine, ethylenediamine, glucoseamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resin But are not limited to, salts thereof. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Crystallization often results in solvates of the compounds of the present invention. The present invention includes all solvates of the disclosed compounds. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of a solvent and one or more molecules of a compound of the invention. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be organic for daily use. Accordingly, the compounds of the present invention may exist as hydrates, including monohydrates, dihydrates, semi-hydrates, sesquihydrates, trihydrates, dihydrates, and the like, as well as the corresponding solvated forms. The compounds of the present invention may be true solvates, while in other cases the compounds of the present invention may only have adventitious water or another solvent, or may be a mixture of water and some negatively charged solvents.

The compounds of the present invention, or salts, tautomers or solvates thereof, may contain one or more asymmetric centers and thus may be present as (R) - or (S) - in the context of absolute stereochemistry, Diastereoisomers, and other stereoisomeric forms which may be defined as - (L) - or (L) -. The present invention is intended to include all such possible isomers, as well as racemic and optically pure forms thereof. Optically active (+) and (-), (R) - and (S) - or (D) - and (L) -isomers can be prepared using chiral synthons or chiral reagents, , &Lt; / RTI &gt; chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include, but are not limited to, chiral synthesis from suitable optically pure precursors or racemates (or racemates of salts or derivatives, for example, using chiral high pressure liquid chromatography Lt; / RTI &gt; Where the compounds described herein contain an olefinic double bond or other centers of geometric asymmetry, and unless otherwise specified, the compounds are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

"Stereoisomer" refers to a compound composed of the same atoms bound by the same bond, but having a different three-dimensional structure, which is not interchangeable. The present invention contemplates a variety of stereoisomers and mixtures thereof, including "enantiomers ", which refers to two stereoisomers that are mirror images in which the molecules can not overlap one another.

"Tautomeric" refers to proton transfer from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any of the above compounds. The various tautomeric forms of the compounds can be readily derivatized by the skilled artisan.

The chemical naming protocol and structural diagrams used herein are prepared using the ACD / Name version 9.07 software program and / or the ChemDraw Ultra version 11.0 software naming program (CambridgeSoft). It is a variant of the nomenclature system. Common names familiar to an ordinary technician are also used.

As mentioned above, in one embodiment of the present invention, compounds useful as fluorescent and / or colored dyes in various assay methods are provided. The compounds have the following structure I, or salts, stereoisomers, or tautomers thereof.

(I)

In this formula,

R ^1a and R ^1b are each independently hydroxyl or alkoxy;

^{R 2a, R 2b, R 2c} , R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R 2q , R ^2r , R ^2s and R ^2t are each independently H, halo or -L ¹ -R ³ ;

R ³ is independently in each case independently selected from the group consisting of an analyte molecule or a hydroxyl, protected hydroxyl, amino, alkylamino, alkoxy, polyalkylether, hydroxylalkoxy, aminoalkoxy, hydroxylpolyalkylether, Substituted with one or more of phosphate, thiophosphate, phospho, thiophospho, phosphoalkyl ether, Ophosphoalkyl ether, thiophosphoalkyl ether, Othiophosphoalkyl ether, phosphoramidite, Alkyl; Or R &lt; ³ &gt; is a microparticle;

L ¹ is an arbitrary linker moiety.

In some other embodiments, the compound has one of the following structures Ia or Ib.

<Formula Ia>

(Ib)

In a further embodiment, the compound has one of the following structures Ic or Id.

<Formula I>

&Lt; EMI ID =

In another embodiment, the compound has one of the following structures Ie or If.

<Formula Ie>

&Lt; RTI ID =

In some of these specific embodiments, the compound has the structure If.

In various embodiments, R &^lt; ^{1a &gt;} or R &lt; ^{1b &gt;} is hydroxyl. In another embodiment, R &^lt; ^{1a &gt;} or R &lt; ^{1b &gt;} is alkoxy. For example, in some embodiments, each of R ^1a and R ^1b is alkoxy.

In some of the embodiments, alkoxy is C ₁ -C ₆ alkoxy, such as methoxy.

In some of any of the above ^{embodiments, R 2a, R 2b, R} 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n At least one of R ^2o , R ^2p , R ^2q , R ^2r , R ^2s or R ^2t is H.

From the other of the ^{embodiments, R 2a, R 2b, R} 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R ^2o , R ^2p , R ^2q , R ^2r , R ^2s or R ^2t is halo. In some more specific embodiments, at least one of R ^2a , R ^2b , R ^2c , R ^2d , R ^2e , R ^2f , R ^2g, or R ^2h is halo, for example, in some embodiments, R ^2b is halo. In some of the above embodiments, halo is bromo.

In various other ^{embodiments, R 2a, R 2b, R} 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o , At least one of R ^2p , R ^2q , R ^2r , R ^2s or R ^2t is -L ¹ -R ³ . For example, in some embodiments, at least one of R ^2a , R ^2b , R ^2c , R ^2d , R ^2e , R ^2f , R ^2g, or R ^2h is -L ¹ -R ³ . In a more specific embodiment, R ^2b is -L ¹ -R ³ .

In another embodiment, R ^2b is -L ¹ -R ³ and ^{R 2a, R 2c, R 2d} , R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R ^2m , R ²ⁿ , R ^2o , R ^2p , R ^2q , R ^2r , R ^2s and R ^2t are H.

In some embodiments, R &lt; ³ &gt; is independently in each instance independently selected from the group consisting of an analyte molecule, or a hydroxyl, protected hydroxyl, amino, alkylamino, alkoxy, polyalkyl ether, hydroxylalkoxy, aminoalkoxy, hydroxylpolyalkylether, Alkyl substituted with one or more of aminopolyalkyl ether, phosphate, thiophosphate, phospho, thiophospho, phosphoalkyl ether, thiophosphoalkyl ether, phosphoramidite or activated phosphorus; Or R ³ is a microparticle. In some of the embodiments, the phosphoalkyl ether is an O-phosphoalkyl ether. In another of the above embodiments, the thiophosphoalkyl ether is an O thiophosphoalkyl ether.

In some of the embodiments, R ³ is independently at each occurrence a hydroxyl, protected hydroxyl, amino, alkylamino, alkoxy, polyalkylether, hydroxylalkoxy, Is alkyl substituted with one or more of ether, phosphate, thiophosphate, phospho, thiophospho, phosphoalkyl ether, thiophosphoalkyl ether, phosphoramidite or activated phosphorus. For example, in some embodiments, R ³ is alkyl substituted with one or more substituents selected from hydroxyl, amino, trityl ether, phosphoramidite, phospho, phosphoalkyl ether, phosphate and polyethylene glycol.

In some of the embodiments, R ³ is independently at each occurrence a hydroxyl, protected hydroxyl, amino, alkylamino, alkoxy, polyalkylether, hydroxylalkoxy, At least one of an ether, a phosphate, a thiophosphate, a phospho, a thiophospho, a phosphoalkyl ether, an ophosphoalkyl ether, a thiophosphoalkyl ether, an O thiophosphoalkyl ether, a phosphoramidite, Substituted alkyl. For example, in some embodiments, R ³ is selected from one or more substituents selected from hydroxyl, amino, trityl ether, phosphoramidite, phospho, phosphoalkyl ether, O phosphoalkyl ether, phosphate and polyethylene glycol Substituted alkyl.

In some embodiments, R &lt; ³ &gt; is alkyl in each case independently substituted with one or more O-phosphoalkyl ethers.

For example, in some of the above embodiments, R ³ is independently in each occurrence independently alkyl substituted with one or more phosphoalkyl ether groups, such as O-phosphoalkyl ether groups, and alkyl ethers of polyalkyl ethers include polyalkyl ethers, Such as ethylene glycol. In some of these embodiments, the alkyl ether of the phosphoalkyl ether is substituted with a substituent selected from hydroxyl and an additional phosphoalkyl ether moiety such as an additional O-phosphoalkyl ether moiety, which is hydroxyl and / .

In another embodiment, R &lt; ³ &gt; is alkyl in each case independently substituted with two O-phosphoalkyl ether groups. In some of these embodiments, the alkyl ether moiety is a polyalkyl ether, such as polyethylene oxide. In some of these embodiments, the alkyl ether moiety is substituted with one or more substituents selected from hydroxyl, amino, and additional O-phosphoalkyl ether moieties, and the alkyl ether of the additional O-phosphoalkyl ether moiety is optionally substituted with one or more substituents selected from the group consisting of hydrogen Lt; / RTI &gt; and / or amino.

Some embodiments of the compounds of structure I are understood to include two or more R &lt; ³ &gt; groups. In such embodiments, the R &lt; ³ &gt; groups may be the same or different. In certain embodiments, the compound of structure I comprises a single R ³ moiety. That ^{is, R 2a, R 2b, R} 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, Only one of R ^2q , R ^2r , R ^2s or R ^2t is -L ¹ -R ³ .

In any of the various other embodiments described above, R &lt; ³ &gt; has one of the following structures.

In this formula,

n ¹ , n ² and n ³ are each independently an integer of 1 to 6;

x and y are each independently an integer of 1 to 10;

In some of the embodiments, n ¹ , n ^2, and n ³ are each 1. In another embodiment, L ¹ is absent and R ³ is directly connected to the remainder of the compound of structure I.

In some other embodiments of the foregoing, x is 5. In another embodiment, y is 5. In some further embodiments, x and y are each 5.

In some different embodiments, R ³ is an analyte molecule, such as a biomolecule. In certain embodiments, R ³ is a biomolecule selected from nucleic acids, amino acids and polymers thereof (eg, DNA, oligonucleotides, proteins, polypeptides, etc.).

In another embodiment, R ³ is a biomolecule and the biomolecule is a nucleic acid, a peptide, a carbohydrate, a lipid, an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an alphamer, an antigen or a prion.

In some different embodiments, R ³ is an analyte molecule selected from a drug, a vitamin, and a small molecule.

In some of the embodiments, the linker L &lt; ¹ &gt; is present. When present, L &lt; ¹ &gt; is suitably selected to provide a covalent attachment between the R &lt; ³ &gt; and the remainder of the compound of structure I. Exemplary L ¹ moieties include, but are not limited to, O, N, S, P and alkylene linkages, and combinations thereof. Suitable L &lt; ^{1 &gt;} groups can be derived by conventional techniques.

In another embodiment, L &lt; ¹ &gt; is absent.

In some embodiments where L &lt; ^{1 &gt;} is present, L &lt; ^{1 &gt;} comprises a phosphate bond to the analyte molecule. In some of these embodiments, L &lt; ¹ &gt;

In the above formula, n ¹ , n ² and n ³ are each independently an integer of 1 to 6. In various other embodiments, n ¹ , n ^2, and n ³ are each 1.

The structure of the compounds of structure I is chosen to optimize the absorbance and / or the emission wavelength. Thus, in various embodiments, the compounds of structure I have a maximum absorbance in the range of about 468 nm to about 508 nm, such as about 478 nm to about 498 nm. In another embodiment, the compound of structure I has a maximum emission in the range of about 495 nm to about 525 nm, for example, about 495 to about 515 nm. For example, in certain embodiments, the dye has a maximum absorbance at about 490 nm and a maximum emission at about 505 nm.

In some more specific embodiments, the compound of Structure I has one of the following structures.

&Lt; Formula 1 >

(2)

(3)

&Lt; Formula 4 >

&Lt; Formula 5 >

(6)

Wherein R ³ is an analyte molecule.

In various other embodiments, the present invention provides an analyte molecule comprising a covalent bond to a compound having the structure I '

<Formula I '

In this formula,

R ^1a and R ^1b are each independently hydroxyl or alkoxy;

^{R 2a, R 2b, R 2c} , R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R 2q , R ^2r , R ^2s and R ^2t are each independently H, halo or -L ¹ -R ³ ;

R ³ is an analyte molecule;

L &^lt; ^{1 &gt;} is an optional linker moiety,

Wherein ^{R 2a, R 2b, R 2c} , R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R At least one of ^2q , R ^2r , R ^2s or R ^2t is -L ¹ -R ³ .

The analyte molecule may be selected from any suitable analyte molecule, including the analyte molecules described herein above.

In various other embodiments, compositions comprising any of the above compounds and one or more biomolecules are provided. In some embodiments, the use of such compositions in an assay method for detecting one or more biomolecules is also provided as described in more detail below.

The arbitrary embodiments of the described compounds of structure (I) as described aspects, and R in the structure (I) as the above-described compound ^1a, R ^1b, R ^2a, R ^2b, R ^2c, R ^2d, R ^2e, R ^2f , to ^{R 2g, R 2h, R 2i} , R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R 2q, R 2r, R 2s, R 2t, L 1 or R ³ variables, It is understood that any specific selection described herein may independently form an embodiment of the present invention that is not specifically described above in combination with other embodiments and / or variables of the compounds of structure &lt; RTI ID = 0.0 &gt; I. &lt; / RTI &gt; In addition, where a list of choices is listed for any particular R or L group in a particular embodiment and / or claim, each individual choice may be removed from the specific embodiment and / or claim, And will be considered to be within the scope of the invention.

It is understood that combinations of substituents and / or variables of the formulas shown in the present description may only be allowed if such a contribution results in a stable compound.

It will also be appreciated by those of ordinary skill in the art that in the methods described herein the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl and the like. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for mercapto include -C (O) -R "wherein R" is alkyl, aryl or arylalkyl, p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acids include alkyl, aryl or arylalkyl esters. The protecting groups can be added or removed according to standard techniques, which are known to the ordinarily skilled artisan and are as described herein. The use of protecting groups is described in detail in Green, T. W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As will be appreciated by the skilled artisan, the protecting group may also be a polymeric resin, such as a Wang resin, a Rink resin or a 2-chlorotrityl-chloride resin.

Furthermore, all compounds of the present invention that are present in the free base or acid form can be converted into their salts by treatment with an appropriate inorganic or organic base or acid by methods known to those of ordinary skill in the art. Salts of the compounds of the present invention can be converted into their free base or acid forms by standard techniques.

The following scheme describes an exemplary method for preparing compounds of the invention, i.e., compounds of structure I.

(I)

^{Wherein, R 1a, R 1b, R} 2a, R 2b, R 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n , R ^2o , R ^2p , R ^2q , R ^2r , R ^2s , R ^2t , R ³ and L ¹ are as defined above.

It is understood that the skilled artisan can prepare these compounds by combining other methods known to the ordinarily skilled artisans or by analogous methods. It will also be appreciated by those of ordinary skill in the art that other compounds of structure I may be prepared in a similar manner as described below, using an appropriate starting component as necessary and altering the parameters of the synthesis, . In general, the starting components may be selected from the group consisting of Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem, USA) or may be obtained from sources such as those known to those of ordinary skill in the art (see for example Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000) Or may be prepared as described herein.

<Reaction Scheme I>

Scheme I illustrates an illustrative method for preparing compounds of structure &lt; RTI ID = 0.0 &gt; I. &lt; / RTI &gt; Referring to Scheme I, the compounds of structures A and B can be prepared by methods well-known to those of ordinary skill in the art. Treatment of two equivalents of A with a strong base such as n-butyl lithium followed by a reaction with B produces the compound of structure I. While Scheme I illustrates the preparation of symmetrical (i. E., The same naphthyl group) compounds of structure I, it is believed that other asymmetric compounds of structure I may be prepared by analogous methods (e. G., Stepwise reactions of differently substituted naphthyls) It will be readily apparent to those of ordinary skill in the art.

In addition, the compounds of structure I obtained by this method can be further modified to obtain different compounds of structure I. For example, in certain embodiments, a compound of structure I comprises at least one -L ¹ -R ³ moiety. In such embodiments, the -L ¹ -R ³ moiety, or its precursor, may be through any widely known method, such as Suzuki coupling. The analyte molecules (e. G., Biomolecules) can be attached via any L ¹ linker by any of a number of conventional methods. For example, modification of the above reaction scheme to include a reactive group capable of forming a covalent bond with a functional group on the analyte molecule is one way to attach the analyte molecule. The reactive groups include, but are not limited to, activated phosphorus compounds (e.g., phosphoramidite), activated esters, amines, alcohols, and the like. Methods for preparing such compounds and methods for reacting them with analyte molecules to form covalent bonds are well known in the art.

In some embodiments, the compound of structure I comprises a covalent bond to an oligonucleotide. Such coupling may be formed by incorporating a phosphoramidite moiety into the compound of structure I and reacting it with an oligomer (or phosphoramidite monomer) under standard DNA synthesis conditions. Methods for DNA synthesis are widely known in the art. Briefly, two alcohol groups are functionalized with a dimethoxytrityl (DMT) group and a 2-cyanoethyl-N, N-diisopropylaminophosphoramidite group, respectively. The phosphoramidite group is coupled to an alcohol group, typically in the presence of an activator such as a tetrazole, and then phosphorus atoms are oxidized to iodine. The dimethoxytrityl group can be removed with an acid (e. G., Chloroacetic acid) to expose the free alcohol and react it with the phosphoramidite group. The 2-cyanoethyl group can be removed after oligomerization by treatment with aqueous ammonia.

The preparation of phosphoramidites used in the oligomerization process is also well known in the relevant art. For example, primary alcohols can be protected as DMT groups by reaction with DMT-Cl. The secondary alcohol is then functionalized as a phosphoramidite by reaction with a suitable reagent such as 2-cyanoethyl N, N-diisopropylchlorophosphoramidite. Methods for the preparation of phosphoramidites and oligomerization thereof are well known in the relevant art and are described in more detail in the Examples.

In another embodiment, the compounds are useful in a variety of analytical methods. For example, in certain embodiments, the present disclosure provides a method of dyeing a sample comprising contacting a sample with a compound of structure I in an amount sufficient to cause an optical reaction when illuminating the sample at an appropriate wavelength, Lt; / RTI &gt;

In another embodiment of the ^{method, R 2a, R 2b, R} 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n , R ^2o , R ^2p , R ^2q , R ^2r , R ^2s or R ^2t is -L ¹ -R ³ , wherein R ³ is an analyte molecule such as a biomolecule. For example, in some embodiments, the biomolecule is a nucleic acid, an amino acid or a polymer thereof (e.g., a polynucleotide or polypeptide). In yet a further embodiment, the biomolecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer or a prion.

In another embodiment of the ^{method, R 2a, R 2b, R} 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n , R ^2o , R ^2p , R ^2q , R ^2r , R ^2s or R ^2t is -L ¹ -R ³ , wherein R ³ is a microparticle. For example, in some embodiments, the microparticles are polymeric beads or non-polymeric beads.

In another embodiment, the optical reaction is a fluorescent reaction.

In another embodiment, the sample comprises cells, and in some embodiments, the cells further include observing the cells by flow cytometry.

In yet a further embodiment, the method further comprises distinguishing the fluorescence response from the fluorescence reaction of the second fluorophore with detectably different optical properties.

In another embodiment,

(a) The compounds of structure I (wherein ^{R 2a, R 2b, R 2c} , R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n , R ^2o , R ^2p , R ^2q , R ^2r , R ^2s, or R ^2t is -L ¹ -R ³ , wherein R ³ is a biomolecule;

(b) detecting the compound by its visual characteristics

A method for detecting a biomolecule visually.

For example, in some embodiments, the biomolecule is a nucleic acid, an amino acid or a polymer thereof (e.g., a polynucleotide or polypeptide). In yet a further embodiment, the biomolecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer or a prion.

In another embodiment, there is provided a method of visually detecting biomolecules,

(a) mixing any of the above compounds with one or more biomolecules;

(b) detecting the compound by its visual characteristics

.

As mentioned above, certain embodiments of the compounds of structure I include analyte molecules (e. G., Biomolecules) or ligands attached thereto (conjugated). Attachment can be by, for example, covalent bonding, ionic bonding, date bonding, hydrogen bonding, and other forms of molecular bonding.

Several types of analyte molecules are suitable for conjugation to compounds of structure I. For example, useful conjugated substrates of the invention include, but are not limited to, compounds of structure I (also referred to herein as "conjugated substrates") comprising analyte molecules attached to a compound of structure I The analyte molecule can be an antigen, small molecule, steroid, vitamin, drug, hapten, metabolite, toxin, environmental contaminant, amino acid, peptide, protein, photosensitizer, nucleotide, oligonucleotide, nucleic acid, carbohydrate, lipid, And non-biological polymers. In one exemplary embodiment, the conjugated substrate is a natural or synthetic amino acid, a natural or synthetic peptide or protein, or an ion-complex moiety. Exemplary peptides include, but are not limited to, protease substrates, protein kinase substrates, phosphatase substrates, neuropeptides, cytokines, and toxins. Exemplary protein conjugates include, but are not limited to, enzymes, antibodies, lectins, glycoproteins, histones, alumin, lipoproteins, avidin, streptavidin, protein A, protein G, casein, picobillery proteins, other fluorescent proteins, hormones, .

The point of attachment of the analyte molecule to the remainder of the compound of structure I will depend on the embodiment and will vary. Also, although the use of a linker is optional and not required in all embodiments, some embodiments include a linker (L ¹ ) between the analyte molecule and the rest of the compound of structure I. It is also contemplated that compounds of structure I may contain more than one analyte molecule. For example, two, three or more than three analyte molecules can be conjugated to the naphthyl and / or anthracene ring of compound (I).

Some methods of linking dyes to various types of analyte molecules are well known in the art. For example, methods for conjugating dyes to analyte molecules are described in R. Haugland, The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 9th Ed., 2002, Molecular Probes, Inc., references; And Brindley, 1992, Bioconjugate Chem. 3: 2), all of which are incorporated herein by reference. For example, a compound of the disclosure may comprise a covalent bond to DNA or RNA via an amide, ester, ether, or thioether linkage via one or more purine or pyrimidine bases; Or attached to a phosphate or carbohydrate by a bond that is an ester, thioester, amide, ether, or thioether. Alternatively, the compound of structure I can be coupled to the nucleic acid by chemical post-modification using, for example, a platinum reagent, or using a photoactivatable molecule such as conjugated psoralen.

The compounds of the present invention are useful in many applications, and for other dyes, see U.S. Patent Nos. 7,172,907; 5,268,486; And U.S. Patent Application No. 20040014981; And 20070042398, all of which patents and applications are incorporated herein by reference in their entirety. For example, fluorescent dyes, such as those described herein, can be used in imaging, such as based on fluorescence detection, including, but not limited to, fluorescence lifetime, anisotropy, mineral oil electron transfer, light fade recovery, . As such, the compounds of structure I can be used in all of the fluorescence-based imaging, microscopy, and spectroscopic techniques and modifications involving such. In addition, they can also be used for broadband curing and in multi-mode imaging. Exemplary fluorescence detection techniques include those that involve detecting fluorescence generated within the system. Such techniques include, but are not limited to, fluorescence microscopy, fluorescence activated cell sorting (FACS), fluorescence flow cytometry, fluorescence correlation spectroscopy (FCS), fluorescence isoelectric hybridization (FISH), multiphoton imaging, diffuse optical tomography, (FIONA) with one nanometer accuracy, free radical-initiated peptide sequence analysis (FRIPs), and second harmonic retinal imaging (SHRIMP) of membrane potential, as well as other methods known in the art , But is not limited thereto.

Alternatively, compounds of structure I can be used as markers or tags to track dynamic behavior in living cells. In this regard, fluorescence recovery (FRAP) after photodegradation is used in combination with the compound of interest to selectively destroy fluorescent molecules within the region of interest with a high intensity laser, The recovery of new fluorescent molecules can be monitored. Variants of FRAP include, but are not limited to, polarized FRAP (pFRAP), fluorescence loss in light-fade (FLIP), and post-fade fluorescence localization (FLAP). Information from FRAP and FRAP variants can be used to determine kinetic properties, including diffusion coefficient, mobility fraction, and transport rate of fluorescently labeled molecules. Methods for such a photo-discoloration based technique are described in Braeckmans, K. et al., Biophysical Journal 85: 2240-2252, 2003; Braga, J. et al., Molecular Biology of the Cell 15: 4749-4760, 2004 ; Haraguchi, T., Cell Structure and Function 27: 333-334, 2002; Gordon, GW et al., Biophysical Journal 68: 766-778, 1995), all of which are incorporated herein by reference in their entirety. do.

Other fluorescence imaging techniques are based on non-radioactive energy transfer reactions, which are a uniform luminescence assay of energy transfer between the donor and acceptor. Such techniques that may utilize the use of the target fluorescent dye include but are not limited to FRET, FET, FP, HTRF, BRET, FLIM, FLI, TR-FRET, FLIE, smFRET, and SHREK. All of these techniques are variants of FRET.

The subject compound may be a biosensor, such as a Ca &lt; ^{2 +} &gt; ion indicator; pH indicator; A phosphorylation indicator, or other indicator, for example, magnesium, sodium, potassium, chloride and halide. For example, biochemical processes often involve protonation and deprotonation of biomolecules with concomitant changes in the pH of the environment. Substituents at the meso-position to the different pH-sensitive groups produce a variety of NIR fluorescent pH sensors with different pKa. To be efficient, substitution at the meso-position will result in a distinct spectral change in response to different pH environments in the fluorophore core and extended? -Conjugation.

The use of the disclosed compounds is by no means limited to analytical methods. In various embodiments, the compounds are used as colorants or dyes in a variety of applications. In this regard, substituents on the core anthracene and / or naphthalene moiety are not particularly limited as long as the compound maintains its desired hue and / or absorbance and / or release properties. The selection of suitable substituents for this purpose is within the skill of the ordinary artisan. Thus, in some embodiments there is provided a compound having the structure I "below, useful as a dye or a colorant, or a salt thereof.

&Lt; RTI ID = 0.0 &gt;

^{Wherein, R 1a, R 1b, R} 2a, R 2b, R 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n , R ^2o , R ^2p , R ^2q , R ^2r , R ^2s and R ^2t are each independently H or a substituent, and the substituents are selected based on the desired hue and / or emission / absorbance characteristics of the compound.

In certain embodiments of the compounds of structure I &quot;, R ^1a and R ^1b are each independently hydroxyl or alkoxy; R ^2a , R ^2b , R ^2c , R ^2d , R ^2e , R ^2f , R ^2g , R ^2h , R ²ⁱ , R ^2j , R ^2k , R ^2l , R ^2m , R ²ⁿ , R ^2o , R ^2p , R ^2q , R ^2r , R ^2s and R ^2t are each independently H, halo or alkyl.

(I. E., Structures I, I 'and / or I " The utility will readily be appreciated by those of ordinary skill in the art for other uses for the disclosed compounds.

The following examples are offered by way of illustration and not by way of limitation.

<Examples>

General method

^& Lt; ¹ &gt; H NMR spectra were obtained on a JEOL 400 MHz spectrometer. ¹ H spectra were referenced for TMS. Reversed phase HPLC dye analysis was performed using a Waters AccuQuid UHPLC system equipped with a 2.1 mm x 50 mm Axuch BEH-C18 column maintained at 45 &lt; 0 &gt; C. Mass spectrum analysis was performed on a Waters / Micromass Quattro micro MS / MS system (MS singleton mode) using MassLynx 4.1 acquisition software. The mobile phase used for LC / MS on the dye was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH 8. Agilent Infinity 1260 with a High Performance Autosampler and a diode array detector using Aapptec ® Spirit ™ Peptide C18 column (4.6 mm x 100 mm, 5 μm particle size) The UHPLC system was used to analyze phosphoramidite and precursor molecules. Tropylium cation injection Improved ionization was used to obtain molecular weights for monomeric intermediates. Excitation and emission profile experiments were recorded on a Cary Eclipse spectrophotometer.

Unless otherwise stated, all reactions were carried out in oven-dried glassware under a nitrogen atmosphere. Commercially available DNA synthesis reagents were purchased from Glen Research (Stirling, Va.). Anhydrous pyridine, toluene, dichloromethane, diisopropylethylamine, triethylamine, acetic acid, pyridine, and THF were purchased from Aldrich. All other chemicals were purchased from Aldrich or TCI and used without further purification.

Example 1

Synthesis of 2-bromo-9,10-bis ((6-methoxynaphthalen-1-yl) ethynyl) anthracene (1)

&Lt; Formula 1 >

A clean dry 200 mL round bottom flask was flushed with nitrogen and dry THF (40 mL). After adding 1- ethynyl-6-methoxynaphthalene (2.0 g, 10.9 mmol), the flask was cooled in an acetone dry ice bath under a nitrogen atmosphere for 30 minutes. Upon cooling, nBuLi 2.5M (4.4 mL, 10.9 mmol) in hexane was added dropwise and the reaction stirred for 1 hr. 2-Bromoanthraquinone (1.05 g, 3.6 mmol) and ether (30 mL) were added, then the reaction was warmed to room temperature and stirred overnight. It was then added a 0.1 M solution (44 mL) of SnCl ₂ in 1M HCl at a time. After 2 h, the reaction mixture was poured into methanol (200 mL) and the resulting slurry was stirred for 1 h. The red solid was isolated by filtration and dried under vacuum (1.15 g, 51%).

The ¹ H NMR spectrum of Compound 1 is shown in FIG. The RP total diode array chromatogram at 215-500 nm of the compound is shown in FIG. The mass spectrum of Compound 1 is shown in FIG.

Example 2

2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) thiophene- Propane-1-ol (2)

(2)

A 250 mL round bottom flask equipped with a condenser was purged with nitrogen and anhydrous THF (50 mL), and then 2 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) Ol (2.2 g, 5.6 mmol). 9-BBN 0.5 M in THF (30.7 mL, 14.0 mmol) was added via syringe and the reaction was heated at reflux for 12 h. After the reaction was cooled to room temperature, 3M K ₂ CO ₃ (2.4 mL) and anhydrous THF (150 mL) were added. Compound 1 (1.74 g, 2.8 mmol) and PdCl ₂ (dppf) (0.41 g, 0.56 mmol) were added and the solution was refluxed for 6 h and then cooled to room temperature over 2 h. The reaction mixture was poured into CH ₂ Cl ₂ (300 mL) and washed with H ₂ O (300 mL). The aqueous layer was back-extracted with additional CH ₂ Cl ₂ (100 mL). The combined washed organic layer with saturated NaCl (300 mL), dried over Na ₂ SO _4, and concentrated in vacuo to give a viscous gum. The isolated crude product wash was then purified by silica gel column chromatography eluting with a gradient of CH ₂ Cl ₂ : hexane (100: 0 v / v) - (0: 100 v / v) Obtained as a solid (1.0 g, 35%). The ¹ H NMR spectrum of Compound 2 is shown in FIG. The total diode array chromatogram at 215-500 nm for compound 2 is shown in FIG. The mass spectrum of Compound 2 is shown in Fig.

Example 3

2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) thiophene- Synthesis of propyl-2-cyanoethyldiisopropylphosphoramidite (3)

(3)

A dry 100 mL round bottom flask was purged with nitrogen and then CH ₂ Cl ₂ (20 mL) and compound 3 (0.20 g, 0.21 mmol) were added. N, N-Diisopropylethylamine (0.18 mL, 10.7 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.15 mL, 0.6 mmol) were added via syringe. After stirring at room temperature for 1 hour, it was determined by TLC analysis that the reaction was complete. The crude reaction mixture was then concentrated in vacuo to give a dark colored gum. The residue was dissolved in acetonitrile, concentrated to dryness and used for dye assembly without further purification.

Example 4

Synthesis of oligonucleotide dyes

Compound 3 was used to prepare additional dye compounds on a 1 μmol scale on an Applied Biosystems 394 DNA / RNA synthesizer. The compound contained a 3'-phosphate group. The dye was synthesized directly on CPG beads or on a polystyrene solid support. The dye was synthesized in the 3 ' to 5 ' direction by standard solid-state DNA method. The coupling method used standard &lt; RTI ID = 0.0 &gt; ss-cyanoethylphosphoramidite &lt; / RTI &gt; All phosphoramidite monomers were dissolved in acetonitrile / dichloromethane (0.1 M solution) and added in successive sequence using the following synthesis cycle: 1) 5'-Dimethoxytri in dichloroacetic acid in toluene 2) coupling of the activator reagent in acetonitrile followed by phosphoramidite, 3) oxidation with iodine / pyridine / water, and 4) acetic anhydride / 1-methylimidazole / Capping with acetonitrile. The synthesis cycle was repeated until the 5'-oligofluorocide was assembled. At the end of the chain assembly, the monomethoxytrityl (MMT) group or the dimethoxytrityl (DMT) group is removed with dichloroacetic acid in dichloromethane or dichloroacetic acid in toluene. The dye was decomposed from the solid support using concentrated aqueous ammonium hydroxide at room temperature for 2-4 hours. The product was concentrated in vacuo and the main product was isolated using a Sephadex G-25 column, yielding RP-HPLC traces (compound 5) as shown in FIG. Figure 8 shows mass spectral data for the dye prepared according to the above procedure (Compound 5). Compound 4 was prepared by coupling compound 3 to a support according to the general procedure described above followed by standard resolution and deprotection.

The structures, spectral characteristics and molecular weights (MW) determined by electrospray mass spectrometry for the exemplary compounds are set forth in Table 1.

Representative intermediates and oligonucleotide dye sequences and their observed mass and optical properties

US patent applications, US patent applications, US patent applications, foreign patents, foreign patent applications, and non-patent applications, including US patent application Ser. No. 61 / 928,147 filed on January 16, 2014, All publications are hereby incorporated by reference to the extent that they are not inconsistent with the description.

From the foregoing, it will be appreciated that various modifications may be made without departing from the spirit and scope of the invention, although the specific embodiments of the invention are described herein for purposes of illustration. Accordingly, the invention is not limited except as by the appended claims.

Claims (45)

A compound having the structure I below, or a salt, stereoisomer or tautomer thereof.
(I)

In this formula,
R ^1a and R ^1b are each independently hydroxyl or C ₁ -C ₆ alkoxy;
^{R 2a, R 2b, R 2c} , R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R 2q , R ^2r , R ^2s and R ^2t are each independently H, halo or -L ¹ -R ³ ;
R ³ is independently in each case independently selected from the group consisting of an analyte molecule or a hydroxyl, protected hydroxyl, amino, alkylamino, alkoxy, polyalkylether, hydroxylalkoxy, aminoalkoxy, hydroxylpolyalkylether, Substituted with one or more of phosphate, thiophosphate, phospho, thiophospho, phosphoalkyl ether, Ophosphoalkyl ether, thiophosphoalkyl ether, Othiophosphoalkyl ether, phosphoramidite, Alkyl; Or R < ³ > is a microparticle;
L &lt; ¹ &gt; is an optional linker moiety;
Wherein ^{R 2a, R 2b, R 2c} , R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R At least one of ^2q , R ^2r , R ^2s, or R ^2t is Is -L ¹ -R ^3. The compound according to claim 1 having one of the following structures Ia, Ib, Ic, Id, Ie or If.
<Formula Ia>

(Ib)

<Formula I>

&Lt; EMI ID =

<Formula Ie>

&Lt; RTI ID =

3. The compound according to claim 2 having the structure If. 4. The method according to any one of claims 1 to 3,
a) R ^1a or R ^1b is hydroxyl;
b) R ^1a or R ^1b is C ₁ -C ₆ alkoxy;
c) R ^1a and R ^1b are C ₁ -C ₆ alkoxy; or
d) R ^1a or R ^1b , or both are C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkoxy is methoxy
compound. 4. The method according to any one of claims 1 to 3,
^{a) R 2a, R 2b,} R 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, At least one of R ^2q , R ^2r , R ^2s, or R ^2t is H;
^{b) R 2a, R 2b,} R 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R ^2q , R ^2r , R ^2s or R ^2t is halo;
c) at least one of R ^2a , R ^2b , R ^2c , R ^2d , R ^2e , R ^2f , R ^2g or R ^2h is halo;
d) R ^2b is halo; or
^{e) R 2a, R 2b,} R 2c, R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R ^2q , R ^2r , R ^2s or R ^2t is halo; At least one of R ^2a , R ^2b , R ^2c , R ^2d , R ^2e , R ^2f , R ^2g or R ^2h is halo; R &^lt; ^2b &gt; is halo, halo is bromo
compound. 4. The method according to any one of claims 1 to 3,
a) at least one of R ^2a , R ^2b , R ^2c , R ^2d , R ^2e , R ^2f , R ^2g or R ^2h is -L ¹ -R ³ ;
b) R ^2b is -L ¹ -R ³ ; or
c) R ^2b is -L ¹ -R ³ ; ^{R 2a, R 2c, R 2d} , R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R 2q, R 2r , R ^2s and R ^2t are H
compound. The method according to claim 6,
a) R ³ is in each occurrence independently selected from the group consisting of hydroxyl, protected hydroxyl, amino, alkylamino, alkoxy, polyalkylether, hydroxylalkoxy, aminoalkoxy, hydroxylpolyalkylether, aminopolyalkylether, Alkyl substituted with one or more of phosphate, phospho, thiophospho, phosphoalkyl ether, Ophosphoalkyl ether, thiophosphoalkyl ether, Othiophosphoalkyl ether, phosphoramidite or activated phosphorus;
b) R ³ is in each case independently selected from the group consisting of hydroxyl, amino, trityl ether, phosphoramidite, phospho, phosphoalkyl ether, O-phosphoalkyl ether, phosphate and polyethylene glycol Substituted alkyl;
c) R ³ is alkyl in each case independently substituted with one or more O-phosphoalkyl ether groups; or
d) R &lt; ³ &gt; is independently in each occurrence one of the following structures
compound.

In this formula,
n ¹ , n ² and n ³ are each independently an integer of 1 to 6;
x and y are each independently an integer of 1 to 10; 7. The compound of claim 6 wherein R &lt; ³ &gt; is independently in each case an analyte molecule. 9. The method of claim 8,
a) the analyte molecule is a biomolecule;
b) the analyte molecule is a biomolecule and the biomolecule is a nucleic acid, an amino acid or a polymer thereof;
c) the analyte molecule is a biomolecule and the biomolecule is a nucleic acid, a peptide, a carbohydrate, a lipid, an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an alphamer, an antigen or a prion; or
d) If the analyte molecule is a drug, vitamin or small molecule
compound. 9. The method of claim 8,
a) L &^lt; ¹ &gt; is present;
b) L ¹ comprises a phosphate bond to the analyte molecule; or
c) L &lt; ^{1 &gt;} has the structure
compound.

In this formula,
n ¹ , n ² and n ³ are each independently an integer of 1 to 6; 2. The compound according to claim 1 having one of the following structures.
&Lt; Formula 1 >

(2)

(3)

&Lt; Formula 4 >

&Lt; Formula 5 >

Wherein R ³ is an analyte molecule. An analyte molecule comprising a covalent bond to a compound having the structure I '
<Formula I '

In this formula,
R ^1a and R ^1b are each independently hydroxyl or C ₁ -C ₆ alkoxy;
^{R 2a, R 2b, R 2c} , R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R 2q , R ^2r , R ^2s and R ^2t are each independently H, halo or -L ¹ -R ³ ;
R ³ is independently in each case an analyte molecule;
L &^lt; ^{1 &gt;} is an optional linker moiety,
Wherein ^{R 2a, R 2b, R 2c} , R 2d, R 2e, R 2f, R 2g, R 2h, R 2i, R 2j, R 2k, R 2l, R 2m, R 2n, R 2o, R 2p, R At least one of ^2q , R ^2r , R ^2s or R ^2t is -L ¹ -R ³ . And adding the analyte molecule of claim 12 to the sample to cause an optical reaction when illuminating the sample at a wavelength in the range of 468 nm to 508 nm. (a) providing a compound of claim 12;
(b) detecting the compound by its visual characteristics
&Lt; / RTI &gt; wherein the analyte molecule is visually detected. (a) mixing a compound of any one of claims 1 to 3, 11, and 12 with one or more biomolecules;
(b) detecting the compound by its visual characteristics
Wherein the biomolecule is detected visually. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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